Method of isolating bile duct progenitor cells

ABSTRACT

The present invention relates to a substantially pure population of viable bile duct progenitor cells, and methods for isolating such cells. The present invention further concerns certain therapeutic uses for such progenitor cells, and their progeny.

BACKGROUND OF THE INVENTION

During the early stages of embryogenesis cells are totipotent and arecapable of multidirectional differentiation. As development proceeds,the totipotent cells become determined and committed to differentiateinto a given specialized cell type. Final differentiation is associatedwith the acquisition of specialized cell functions. Thus, thedifferentiated somatic cells maintain their specialized featuresthroughout the life span of the organism, probably through sustainedinteractions between the genome and its microenvironment and cell-cellinteractions (DiBerardino et al. 1984, Science 224: 946-952; Wetts andFraser, 1988, Science 239: 1142-1144; Fisher, 1984, PNAS 81: 4414-4418).

Because of the tremendous potential of progenitor cells to differentiateinto distinct lineages, there has always existed a need for a continuoussource of these isolated pluripotent progenitor cells. The pluripotentprogenitor cells could be extremely useful in the treatment of differentdisorders that are characterized by insufficient or abnormal functioningof the fully differentiated cells in a given organ, as for example inthe human pancreas or liver.

SUMMARY OF THE INVENTION

The present invention relates to substantially pure preparations ofviable progenitor cells and methods for isolating such cells. Thepresent invention further concerns certain uses for such progenitorcells and their progeny.

In general, the invention features a cellular composition including, asthe cellular component, a substantially pure population of viable bileduct progenitor cells which progenitor cells are capable ofproliferation in a culture medium. In a preferred embodiment, thecellular composition has fewer than about 20%, more preferably fewerthan about 10%, most preferably fewer than about 5% of lineage committedcells.

In general, the progenitor cells of the present invention areproliferative cells which can differentiate into cells making up thetissues of the gut, e.g., the liver, pancreas, gallbladder, intestines,etc. That is, the progenitor cells can give rise to differentiated cellsof hepatic, pancreatic, gallbladder or intestinal lineages. However, asdescribed in the pending examples, the subject method can be used toisolate populations of hematopoietic stem/progenitor cells. In preferredembodiments, the subject progenitor cells are pluripotent, e.g., theprogenitor cells are capable of differentiating into two or moredistinct lineages.

In one embodiment, the progenitor cells of the present invention arecharacterized by an ability for self-regeneration in a culture mediumand differentiation to pancreatic lineages. In a preferred embodiment,the progenitor cells are inducible to differentiate into pancreaticislet cells, e.g., β islet cells, α islet cells, δ islet cells, or φislet cells. Such pancreatic progenitor cells may be characterized incertain circumstances by the expression of one or more of: homeodomaintype transcription factors such as STF-1; PAX gene(s) such as PAX6;PTF-1; hXBP-1; HNF genes(s); villin; tyrosine hydroxylase; insulin;glucagon; and/or neuropeptide Y.

In another embodiment, the invention features progenitor cells, such asmay be obtained from a non-hepatic bile duct according to the presentinvention, which are characterized by an ability to differentiate (e.g.,by induction) into hepatocytes when maintained in culture. Exemplaryhepatic progenitor cells may be characterized by the expression of oneor more of: a hepatocyte nuclear factor (HNF) transcription factor,e.g., HNF1α, HNF1β, HNF3β, HNF3γ, and/or HNF4; ATBF1; AFP; a LIM typehomeobox gene such as Islet-1; a “forkhead” transcription factor, suchas fkh-1; a CCAAT-enhancer binding protein (C/EBP), such as C/EBP-β;oval cell marker OV-6; cytokeratins (such as type 7 or 19), mucinSpan-1; OC.2; OC.3; and/or γ-glutamyl transpeptidase.

In still another embodiment, the invention features hematopoieticprogenitor cells which may differentiate into one or more of anerythrocyte, a megakaryocyte, a monocyte, a granulocyte and/or aneosinophils as well as fully differentiated lymphoid cells such as Blymphocytes and T lymphocytes. Certain of the subject hematopoieticprogenitor cells may be characterized by expression of such markers asc-kit, CD34 and/or CD33, as well as OV-6. CAM5.2 and/or cytokeratin type7 or 18.

In yet another embodiment, the invention features a pharmaceuticalcomposition including as the cellular component a substantially purepopulation of viable bile duct progenitor cells, which progenitor cellsare capable of proliferation in a culture medium.

In general, the preferred progenitor cells will be of mammalian origin,e.g., cells isolated from a primate such as a human, from a miniatureswine, or from a transgenic mammal, or are the cell culture progeny ofsuch cells.

In preferred embodiments, the subject progenitor cells can be maintainedin cell/tissue culture for at least about 7 days, more preferably for atleast about 14 days, most preferably for at least about 21 days orlonger.

In another aspect, the invention features a cellular compositioncomprising, as a cellular population, at least 75% (though morepreferably at least 80, 90 or 95%) progenitor cells isolated from a bileduct and capable of self-regeneration in a culture medium.

In yet another aspect, the invention features, a cellular compositionconsisting essentially of, as the cellular population, viablenon-hepatic duct progenitor cells capable of self-regeneration in aculture medium and differentiation to members of the hepatic, pancreaticand gallbladder lineages. For instance, in certain embodiments theprogenitor cells are isolated from cystic duct explants, pancreatic ductexplants common bile duct explants, or are the cell culture progeny ofsuch cells.

Another aspect of the invention features a method for isolatingprogenitor cells from a bile duct. In general, the method provides forculturing an isolated population of cells having a microarchitecture ofa mammalian bile duct, e.g. a micro-organ explant in which the originalepithelial-mesenchymal microarchitecture is maintained, wherein thedimensions of the explant provide the isolated population of cells asmaintainable in culture for at least twenty-four hours, and includes inthe population of cells at least one progenitor cell which canproliferate under such culture conditions. The cultured cell populationis contacted with an agent, e.g., a mitogenic agent such as a growthfactor which agent causes proliferation of progenitor cells in thecultured population. Subsequently, progenitor cells from the explantthat proliferate in response to the agent are isolated, such as bydirect mechanical separation of newly emerging buds from the rest of theexplant or by dissolution of all or a portion of the explant andsubsequent isolation of the progenitor cell population.

In another preferred embodiment, the agent is a growth factor. e.g., thegrowth factor is selected from a group consisting of IGF, TGF, FGF, EGF,HGF, or VEGF. In other embodiments, the growth factor is a member of theTGFβ superfamily, preferably of the DVR (dpp and vgl related) family,e.g., BMP2 and/or BMP7.

In another preferred embodiment, the population of cells is cultured ina medium deficient in biological extracts. e.g., deficient in serum.

In a preferred embodiment, the bile duct is a common bile duct.

In another aspect, the invention features, a method for screening acompound for ability to modulate one of growth, proliferation, and/ordifferentiation of progenitor cells obtained from a bile duct,including: (i) establishing an isolated population of cells having amicroarchitecture of a mammalian bile duct, e.g., a micro-organ explantin which the original epithelial-mesenchymal microarchitecture ismaintained, wherein the dimensions of the explant provide the isolatedpopulation of cells as maintainable in culture for at least twenty-fourhours, and includes at least one progenitor cell which has the abilityto proliferate in the culture; (ii) contacting the population of cellswith a test compound; and (iii) detecting one of growth, proliferation,and/or differentiation of the progenitor cells in the population,wherein a statistically significant change in the extent of one ofgrowth, proliferation, and/or differentiation in the presence of thetest compound relative to the extent of one of growth, proliferation,and/or differentiation in the absence of the test compound indicates theability of the test compound to modulate one of the growth,proliferation, and/or differentiation.

In another aspect the invention features, a method for treating adisorder characterized by insufficient insulin activity, in a subjectincluding introducing into the subject a pharmaceutical compositionincluding pancreatic progenitor cells, or differentiated cells arisingtherefrom and a pharmaceutically acceptable carrier.

In a preferred embodiment the subject is a mammal, e.g., a primate,e.g., a human.

In another preferred embodiment the disorder is an insulin dependentdiabetes, e.g., type I diabetes.

In yet another preferred embodiment, the pancreatic progenitor cells areinduced to differentiate into pancreatic islet cells, e.g., β isletcells, α islet cells, δ islet cells, or φ islet cells, subsequent tobeing introduced into the subject. Preferably, the pancreaticprogenitors cells are induced to differentiate into pancreatic islet,e.g., β islet cells, α islet cells, δ islet cells or φ islet cells, inculture prior to introduction into the subject.

In another aspect, the invention features, a method for treating adisorder characterized by insufficient liver function, in a subjectcomprising introducing into the subject a pharmaceutical compositionincluding hepatic progenitor cells and a pharmaceutically acceptablecarrier.

In a preferred embodiment, the subject is a mammal, e.g., a primate,e.g., a human.

In another preferred embodiment the disorder is selected from the groupconsisting of cirrhosis, hepatitis B, hepatitis C, sepsis, or ELAD. Inyet another preferred embodiment, the hepatic progenitor cells areinduced to differentiate into hepatocytes subsequent to being introducedinto the subject. Preferably, the hepatic progenitors cells are inducedto differentiate into hepatocytes in culture prior to introduction intothe subject.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare described in the literature. See, for example, Molecular Cloning ALaboratory Manual; 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D.Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press. Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds. Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, eds. 1986); Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press. Cold Spring Harbor. N.Y. 1986).

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting average number of BrdU positive nuclei in acommon bile duct explant, 24 hours after administration of a growthfactor. Growth factors, EGF, TGF-αand basic FGF (bFGF) were administeredin three doses: 1 ng/ml, 10 ng/ml and 100 ng/ml. DMEM minimal media wasused as a control. Average fluorescence intensity was determined basedon the number of positive nuclei.

FIG. 2 is a graph depicting the percentage of BrdU labeled positivecells in a common bile duct explant, 24 hours after administration of agrowth factor. Growth factors. EGF, TGF-α, and bFGF were administeredand DMEM minimal media was used as a control.

FIG. 3 is a micrographs depicting in vitro growth and expansion of thecells within a common bile duct explant in response to administration ofgrowth factor bFGF.

FIGS. 4A-4C a micrographs (120×) illustrating the types of cellsexpanded from bile duct microorgan cultures by IGF-1 stimulation. Theaddition of IGF-1 at 100 ng/ml resulted in at least three types ofresponses; no growth or change in morphology (FIG. 4A); the appearanceof “spiny” colonies which are termed “N-type” colonies due to theneurite-like appearance of the colony extensions (FIG. 4B); and theappearance of “L-type” colonies for liver-like, due to the epithelialblebs and formation of red blood cell foci (FIG. 4C) which wereidentical in appearance to cultured embryonic liver (not shown).

FIG. 5 is a graph demonstrating that ductal microorgan cultures onmatrigel can give riase to multiple colony types.

DETAILED DESCRIPTION OF THE INVENTION

The ability to isolate distinct populations of progenitor cells has beenan important problem in modern biology. It can be easily envisioned thatsuch isolated pluripotent progenitor cells could be very useful fortreatment of various disorders associated with loss or abnormalfunctioning of fully differentiated cells in a given organ. For example,the ability to introduce isolated progenitor cells capable of subsequentdifferentiation, either in culture or when introduced into a subject,into functional islet cells, would have important implications for thetreatment of insulin-dependent diabetes. In the same manner, the abilityto deliver purified hepatic progenitor cells, having the ability todifferentiate into mature hepatocytes, could be potentially useful inthe process of liver regeneration or for treatment of disorderscharacterized by insufficient liver function. Prior to the instantinvention, there was no apparently reliable source of purifiedpopulations of progenitor cells from gut tissue capable of furtherdifferentiation into distinct pancreatic, hepatic, gallbladder,intestinal or hematopoietic lineages, either in culture or whenintroduced into a subject.

Accordingly, certain aspects of the present invention relate to isolatedpopulations of progenitor cells capable of subsequent differentiation todistinct pancreatic, hepatic, gallbladder, intestinal or hematopoieticlineages, methods for isolating such cells and therapeutic uses for suchcells.

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The term “culture medium” is recognized in the art, and refers generallyto any substance or preparation used for the cultivation of livingcells. Accordingly, a “tissue culture” refers to the maintenance orgrowth of tissue, e.g., explants of organ primordia or of an adult organin vitro so as to preserve its architecture and function. A “cellculture” refers to a growth of cells in vitro; although the cellsproliferate they do not organize into tissue per se.

Tissue and cell culture preparations of the subject micro-organ explantsand amplified progenitor cell populations can take on a variety offormats. For instance, a “suspension culture” refers to a culture inwhich cells multiply while suspended in a suitable medium. Likewise, a“continuous flow culture” refers to the the cultivation of cells orductal explants in a continuous flow of fresh medium to maintain cellgrowth. e.g. viablity. The term “conditioned media” refers to thesupernatant, e.g. free of the cultured cells/tissue, resulting after aperiod of time in contact with the cultured cells such that the mediahas been altered to include certain paracrine and/or autocrine factorsproduced by the cells and secreted into the culture.

The terms “explant” and “micro-organ explant” refer to a portion of anorgan taken from the body and grown in an artificial medium.

The term “tissue” refers to a group or layer of similarly specializedcells which together perform certain special functions.

The term “organ” refers to two or more adjacent layers of tissue whichlayers of tissue maintain some form of cell-cell and/or cell-matrixinteraction to form a microarchitecture.

The term “lineage committed cell” refers to a progenitor cell that is nolonger pluripotent but has been induce to differentiate into a specificcell type, e.g., a pancreatic, hepatic or intestinal cell.

The term “progenitor cell” refers to an undifferentiated cell which iscapable of proliferation and giving rise to more progenitor cells havingthe ability to generate a large number of mother cells that can in turngive rise to differentiated, or differentiable daughter cells. As usedherein, the term “progenitor cell” is also intended to encompass a cellwhich is sometimes referred to in the art as a “stem cell”. In apreferred embodiment, the term “progenitor cell” refers to a generalizedmother cell whose descendants (progeny) specialize, often in differentdirections, by differentiation, e.g., by acquiring completely individualcharacters, as occurs in progressive diversification of embryonic cellsand tissues. A “bile duct progenitor cells” refers to progenitor cellsarising in tissue of a bile duct and giving rise to such differentiatedprogeny as, for example, hepatic, pancreatic, intestinal, gallbladder orhematopoietic lineages.

As used herein the term “bile duct” refers to an intricate system ofducts, e.g., generally tubular structures used for secretion, eitherneonatal or adult. The term includes the hepatic duct, cystic duct, andpancreatic duct. The term “pancreatic duct” includes the accessorypancreatic duct, dorsal pancreatic duct, main pancreatic duct andventral pancreatic duct. The term bile duct also encompasses the commonbile duct. The main function of the bile duct is to allow bile and othermaterials to drain from these organs and enter the gastrointestinaltract.

As used herein the term “common bile duct” refers to a region of thebile duct either adult or neonatal, originating from the liver bilecanaliculi and extending down to the papilla of Vater at the duodenaljunction. The common bile duct is continuous with hepatic, cystic andcertain pancreatic ducts.

As used herein the term “non-hepatic bile duct” refers to that bile ducttissue which is not hepatic duct tissue, e.g., those portions of thecommon bile duct posterior to the hepatic duct. In addition, the termincludes cystic and pancreatic ducts.

As used herein the term “substantially pure”, with respect to progenitorcells, refers to a population of progenitor cells that is at least about75%, preferably at least about 85%, more preferably at least about 90%and most preferably at least about 95% pure with respect to progenitorcells making up a total cell population. Recast, the term “substantiallypure” refers to a population of progenitor cell of the present inventionthan contain fewer than about 20%, more preferably fewer than about 10%,most preferably fewer than about 5%, of lineage committed cells in theoriginal unamplified and isolated population prior to subsequentculturing and amplification.

As used herein the term “cellular composition” refers to a preparationof cells, which preparation may include, in addition to the cellsnon-cellular components such as cell culture media, e.g. proteins, aminoacids, nucleic acids, nucleotides, co-enzyme, anti-oxidants, metals andthe like. Furthermore, the cellular composition can have componentswhich do not affect the growth or viability of the cellular component,but which are used to provide the cells in a particular format, e.g., aspolymeric matrix for encapsulation or a pharmaceutical preparation.

As used herein the term “animal” refers to mammals, preferably mammalssuch as humans. Likewise, a “patient” or “subject” to be treated by themethod of the invention can mean either a human or non-human animal.

As described below, in a preferred embodiment, the progenitor cells ofthe present invention are pancreatic or hepatic progenitor cells. Theterm “pancreas” is art recognized, and refers generally to a large,elongated racemose gland situated transversely behind the stomach,between the spleen and duodenum. The pancreatic exocrine function, e.g.,external secretion, provides a source of digestive enzymes. Indeed,“pancreatin” refers to a substance from the pancreas containing enzymesprincipally amylase, protease, and lipase, which substance is used as adigestive aid. The exocrine portion is composed of several serous cellssurrounding a lumen. These cells synthesize and secrete digestiveenzymes such as trypsinogen, chymotrypsinogen, carboxypeptidase,ribonuclease, deoxyribonuclease, triacylglycerol lipase phospholipaseA₂, elastase, and amylase.

The endocrine portion of the pancreas is composed of the islets ofLangerhans. The islets of Langerhans appear as rounded clusters of cellsembedded within the exocrine pancreas. Four different types of cells—α,β, δ, and φ—have been identified in the islets. The α cells constituteabout 20% of the cells found in pancreatic islets and produce thehormone glucagon. Glucagon acts on several tissues to make energyavailable in the intervals between feeding. In the liver, glucagoncauses breakdown of glycogen and promotes gluconeogenesis from aminoacid precursors. The δ cells produce somatostatin which acts in thepancreas to inhibit glucagon release and to decrease pancreatic exocrinesecretion. The hormone pancreatic polypeptide is produced in the φcells. This hormone inhibits pancreatic exocrine secretion ofbicarbonate and enzymes, causes relaxation of the gallbladder, anddecreases bile secretion. The most abundant cell in the islets,constituting 60-80% of the cells, is the β cell, which produces insulin.Insulin is known to cause the storage of excess nutrients arising duringand shortly after feeding. The major target organs for insulin are theliver, muscle, and fat-organs specialized for storage of energy.

The term “pancreatic progenitor cell” refers to a cell which candifferentiate into a cell of pancreatic lineage, e.g. a cell which canproduce a hormone or enzyme normally produced by a pancreatic cell. Forinstance, a pancreatic progenitor cell may be caused to differentiate,at least partially, into α, β, δ, or φ islet cell or a cell of exocrinefate. The pancreatic progenitor cells of the invention can also becultured prior to administration to a subject under conditions whichpromote cell proliferation and differentiation. These conditions includeculturing the cells to allow proliferation and confluence in vitro atwhich time the cells can be made to form pseudo islet-like aggregates orclusters and secrete insulin, glucagon, and somatostatin.

The term “liver” refers to the large, dark-red gland in the upper partof the abdomen on the right side, just beneath the diaphragm. Itsmanifold functions include storage and filtration of blood, secretion ofbile, conversion of sugars into glycogen, and many other metabolicactivities.

The liver is a gland that supplies bile to intestine. In adultvertebrates, this function is a minor one, but the liver originallyarose as a digestive gland in lower chordates. Throughout the liver, anetwork of tiny tubules collects bile—a solution of salts, bilirubin(made when hemoglobin from red blood cells is broken down in liver) andfatty acids. Bile accumulates in the gall bladder, which empties intothe small intestine by way of a duct. Bile has two functions in theintestine. First, it acts as a detergent, breaking fat into smallglobules that can be attacked by digestive enzymes. Second, and moreimportant, bile salts aid in the absorption of lipids form theintestine; removal of the gall bladder sometimes causes difficulty withlipid absorption.

Digested food molecules absorbed into the bloodstream from the intestinepass directly to the liver by way of the hepatic portal vein. Beforethese molecules pass on into the rest of the body, the liver may changetheir concentration and even their chemical structure. The liverperforms a vital role in detoxifying otherwise poisonous substances. Inaddition, it stores food molecules that reach it form the intestine,converts them biochemically, and releases them back into the blood at acontrolled rate. For instance, the liver removes glucose from the bloodunder the influence of the hormone insulin and stores it as glycogen.When the level of glucose in the blood falls, the hormone glucagoncauses the liver to break down glycogen and release glucose into theblood.

The liver also synthesizes many of the blood proteins (e.g., albumins)and releases them into the blood when they are needed. In addition, theliver converts nitrogenous wastes into the form of urea for excretion bythe kidneys. With the kidneys, the liver is vital in regulating what theblood contains when it reaches all the other organs of the body. Becausethe liver is the body's major organ for making all these biochemicaladjustments severe liver damage or loss of the liver is rapidly fatal.

The term “hepatic progenitor cell” as used herein refers to a cell whichcan differentiate in a cell of hepatic lineage, such a liver parenchymalcell. e.g., a hepatocyte. Hepatocytes are some of the most versatilecells in the body. Hepatocytes have both endocrine and exocrinefunctions and synthesize and accumulate certain substance, detoxifyothers and secrete others to perform enzymatic, transport, or hormonalactivities. The main activities of liver cells include bile secretion,regulation of carbohydrate lipid, and protein metabolism, storage ofsubstances important in metabolism; degradation and secretion ofhormones, and transformation and excretion of drugs and toxins. Thehepatic progenitor cells of the invention can also be cultured prior toadministration to a subject under conditions which promote cellproliferation and differentiation.

The term “hematopoietic cells” herein refers to fully differentiatedmyeloid cells such as erythrocytes or red blood cells, megakaryocytes,monocytes, granulocytes, and eosinophils, as well as fullydifferentiated lymphoid cells such as B lymphocytes and T lymphocytes.Thus, a hematopoietic stem/progenitor cell includes the varioushematopoietic precursor cells from which these differentiated cellsdevelop, such as BFU-E (burst-forming units-erythroid), CFU-E (colonyforming unit-erythroid), CFU-Meg (colony forming unit-megakaryocyte),CFU-GM (colony forming unit-granulocyte-monocyte), CFU-Eo (colonyforming unit-eosinophil), and CFU-GEMM (colony formingunit-granulocyte-erythrocyte-megakaryocyte-monocyte).

Certain terms being set out above, it is noted that one aspect of thepresent invention features a method for isolating progenitor cells frommicro-organ explants, e.g., ductal tissue explants. A salient feature ofthe subject method concerns the use of defined explants as sources fromwhich discrete progenitor cell populations can be amplified. Forinstance, as described below, the progenitor source ductal tissueexplants preferably are derived with dimensions that allow the explantedtissue to maintain its microarchitecture and biological function forprolonged periods of time in culture, e.g., the dimensions of theexplant preserve the normal tissue architecture and at least a portionof the normal tissue function that is present in vivo. Such tissueexplants can be maintained, for instance in minimal culture media forextended periods of time (e.g., for 21 days or longer) and can becontacted with different factors. Accordingly, carefully definedconditions can be acquired in the culture so as selectively activatediscrete populations of cells in the tissue explant. The progenitorcells of the present invention can be amplified, and subsequentlyisolated from the explant, based on a proliferative response upon, forexample, addition of defined growth factors or biological extracts tothe culture.

In general, the method of the present invention can be used to isolateprogenitor cells from a bile duct explant by steps beginning with theculturing of an isolated population of cells having a microarchitectureof a mammalian bile duct, e.g. a micro-organ explant in which theoriginal epithelial-mesenchymal microarchitecture of the originatingduct is maintained, wherein the dimensions of the explant provide theisolated population of cells as maintainable in culture for at leasttwenty-four hours and includes in the population of cells at least oneprogenitor cell which can proliferate under such culture conditions. Theexplant is contacted with an agent. e.g., a mitogenic agent such as agrowth factor or other biological extract, which agent causesproliferation of progenitor cells in the cultured population.Subsequently, progenitor cells from the explant that proliferate inresponse to the agent are isolated, such as by direct mechanicalseparation from the rest of the explant or by dissolution of all or aportion of the explant and subsequent isolation of the progenitor cellpopulation.

In a illustrative embodiment, the size of the particular ductal explantwill depend largely on (i) the availability of tissue, and (ii) a needfor similar availability of nutrients to all cells in the tissue bydiffusion. In a preferred embodiment, the ductal tissue explant isselected to provide diffusion of adequate nutrients and O₂ to every cellin a three dimensional organ. Accordingly, the size of the explant isdetermined by the requirement for a minimum level of accessibility toeach cell absent specialized delivery structures or syntheticsubstrates.

A salient feature of the micro-organ cultures used to in the subjectmethods, according to the invention, is the ability to preserve thecellular microenvironment found in vivo for a ductal tissue. Theinvention is based, in part, upon the discovery that under certaincircumstances growth of cells of both a stromal and epithelial layer,provided together in the same explant, will sustain active proliferationof cells of each layer. Moreover, the cell-cell and cell-matrixinteractions provided in the explant itself are sufficient to supportcellular homeostasis. e.g., maturation, differentiation and segregationof cells in the explant culture, thereby sustaining themicroarchetecture and function of the explant for prolonged periods oftime.

An example of physical contact between a cell and a noncellularsubstrate (matrix) is the physical contact between an epithelial celland its basal lamina. An example of physical contact between a cell andanother cell includes actual physical contact maintained by, for,example, intercellular cell junctions such as gap junctions and tightjunctions. Examples of functional contact between one cell and anothercell includes electrical or chemical communication between cells. Inaddition, many cells communicate with other cells via chemical messages,e.g., hormones, which either diffuse locally (paracrine signalling andautocrine signalling), or are transported by the vascular system to moreremote locations (endocrine signalling).

Not wishing to be bound by any particular theory, this microarchitectureof the ductal explants can be extremely important for the maintenance ofthe explant in minimal media, e.g.; without exogenous sources of serumor growth factors, as the ductal explants can apparently be sustained insuch minimal media by paracrine factors resulting from specific cellularinteractions within the sample. Moreover, there is also a possibilitythat certain growth factors might act indirectly by activating cellsother than progenitor cells, to produce mitogenic factors thatsubsequently cause proliferation of progenitor cells within the explant.Accordingly, the ductal explants are derived such that they compriseboth an epithelial layer and a stromal layer and maintain in vitro thephysical and/or functional interaction between these two component ofthe explant.

However, the phrase “maintain, in vitro, the physical and/or functionalinteraction” is not intended to exclude an isolated population of cellsin which at least one cell develops physical and/or functional contactwith at least one cell or noncellular substance with which it is not inphysical and/or functional contact in vivo. An example of such adevelopment is of course proliferation of at least one cell of theisolated population of cells.

As emphasized through the present application, the micro-organ culturesused to prepare progenitor cells according to the invention preserve thenormal tissue architecture that is present in vivo, e.g., the originalepithelial-mesenchymal organization. In preferred embodiments thepopulations of cells of the ductal explants are grouped in a manner thatpreserves the natural affinity of one cell to another, e.g., to preservelayers of different cells present in explant. Such an associationfacilitates intercellular communication. Many types of communicationtakes place among animal cells. This is particularly important indifferentiating cells where induction is defined as the interactionbetween one (inducing) and another (responding) tissue or cell, as aresult of which the responding cells undergo a change in the directionof differentiation. Moreover, inductive interactions occur in embryonicand adult cells and can act to establish and maintain morphogeneticpatterns as well as induce differentiation (Gurdon (1992) Cell 68:185-199). Accordingly, an exemplary micro-organ cultures prepared inaccordance to use in the progenitor amplification method of theinvention are described in Example 1 and include a epithelial andmesenchymal cells grouped in a manner that includes a plurality oflayers so as to preserve the natural affinity and interaction of onecell to another in and between each layer.

In addition to isolating a ductal explant which retains the cell-celland cell-matrix architecture of the originating duct, the dimensions ofthe explant are important to the viability of the cells therein, e.g.,where the micro-organ culture is intended to be sustained for prolongedperiods of time, e.g., 7-21 days or longer. Accordingly, the dimensionsof the explant are selected to provide diffusion of adequate nutrientsand gases (e.g., O₂, CO₂, etc) to every cell in the three dimensionalmicro-organ explant as well as diffusion of cellular waste out of theexplant so as to minimize cellular toxicity and concommitant death dueto localization of the waste in the micro-organ. Thus, in addition tothe requirement of both epithelial and mesenchymal components the sizeof the explant is determined by the requirement for a minimum level ofaccessibility to each cell in the absence specialized deliverystructures or synthetic substrates. As described herein, thisaccessibility can be maintained if Aleph, an index calculated from thethickness and the width of the explant is at least greater thanapproximately 1.5 mm⁻¹. As used herein, a surface to area index, “Aleph”is defined as Aleph=1/x+1/a>1.5 mm⁻¹; wherein x=radial thickness anda=the axial length of the duct explant in millimeters. Accordingly, thepresent invention provides that the surface area to volume index of thetissue explant is maintained within a selected range. This selectedrange of surface area to volume indice provides the cells access tonutrients and to avenues of waste disposal by diffusion in a mannersimilar to cells of the in vivo organ from which the explant originated.

Examples of Aleph are provided in Table I wherein for example, anexplant having a thickness (x) of 0.1 mm and a width (a) of 1 mm wouldhave an Aleph index of 11. In another instance, if x=0.3 mm and a=4 mm,the Aleph is 3.48 mm⁻¹. To further illustrate, Applicant has observedthat when x is varied and a is constant at 4 mm, the proliferativeactivity of cells in a cultured explant is substantially reduced as thethickness of the explant increases. Accordingly, at 900 μm thickness,the number of proliferating cells in a micro-organ culture was found toabout 10 fold less than in tissue from a similar source having athickness of 300 μm. The Aleph index for a tissue having a thickness of900 μm is 1.36 mm⁻¹, below the minimum described herein whereas theAleph index for tissue having a thickness of 300 μm is 3.58 mm⁻¹ whichis well within the range defined herein. TABLE I Different values forthe surface area to volume ratio index “Aleph”, as a function of a(width) and x (thickness) in mm⁻¹

Again, not wishing to be bound by any particular theory, a number offactors provided by the three-dimensional culture system may contributeto its success in the subject method of activating progenitor cellpopulations:

(a) The appropriate choice of the explant size, vis-á-vis the use of theabove Aleph calculations, three-dimensional matrix provides appropriatesurface area to volume ratio for adequate diffusion of nutrients to allcells of the explant, and adequate diffusion of cellular waste away fromall cells in the explant.

(b) Because of the three-dimensionality of the matrix, various cellscontinue to actively grow, in contrast to cells in monolayer cultures,which grow to confluence, exhibit contact inhibition, and cease to growand divide. The elaboration of growth and regulatory factors byreplicating cells of the explant may be partially responsible forstimulating proliferation and regulating differentiation of cells inculture.

(c) The three-dimensional matrix retains a spatial distribution ofcellular elements, e.g., the epithelial-mesenchymal micoarchitecturewhich closely approximates that found in the counterpart tissue in vivo.

(d) The cell-cell and cell-matrix interactions may allow theestablishment of localized microenvironments conducive to cellularinduction and/or maturation. It has been recognized that maintenace of adifferentiated cellular phenotype requires not onlygrowth/differentiation factors but also the appropriate cellularinteractions. The present invention effectively mimics themicroenvironment. Accordingly, the micro-organ preserves interactionswhich may be required to maintain the cells supporting the progenitorcells, the cells (if any) providing inductive signals to the progenitorcells, and the progenitor cells themselves.

To further illustrate, the appended examples demonstrate that microorganexplants, in which the original epithelial-mesenchymal microarchitectureof the common bile duct are prepared by transverse sectioning of theduct every 300 μm, can be maintained in simple media (DMEM) for severalweeks and can be used to isolate progenitor cells of the presentinvention by growth induction upon contact with certain growth factors.

There are a large number of tissue culture media that exist forculturing tissue from animals. Some of these are complex and some aresimple. While it is expected that the ductal explants may grow incomplex media, it will generally be preferred that the explants bemaintained in a simple medium, such as Dulbecco's Minimal EssentialMedia (DMEM), in order to effect more precise control over theactivation of certain progenitor populations in the explant.Furthermore, although the cultures may be grown in a media containingsera or other biological extracts such as pituitary extract, it has beendemonstrated that neither sera nor any other biological extract isrequired for explants derived according to the above considerations (seeU.S. Ser. No. 08/341,409,). Moreover, the explants can be maintained inthe absence of sera for extended periods of time. In preferredembodiments of the invention, the growth factors or other mitogenicagents are not included in the primary media for maintenance of thecultures in vitro, but are used subsequently to cause proliferation ofdistinct populations of progenitor cells. See the appended examples.

The tissue explants may be maintained in any suitable culture vessel,such as a 12 or 24 well microplate, and may be maintained under typicalculture conditions for cells isolated from the same animal. e.g., suchas 37° C. in 5% CO₂. The cultures may be shaken for improved aeration,the speed of shaking being, for example, 12 rpm.

In order to isolate progenitor cells from the ductal explants, it willgenerally be desirable to contact the explant with an agent which causesproliferation of one or more populations of progenitor cells in theexplant. For instance, a mitogen, e.g., a substance that induces mitosisand cell transformation can be used to detect a progenitor cellpopulation in the explant and where desirable, to cause theamplification of that population. To illustrate, a purified orsemi-purifed preparation of a growth factor can be applied to theculture. Induction of progenitor cells which respond to the appliedgrowth factor can be detected by proliferation of the progenitor cells.However, as described below, amplification of the population need notoccur to a large extent in order to use certain techniques for isolatingthe responsive population.

In yet other embodiments, the ductal explants and/or amplifiedprogenitor cells can be cultured on feeder layers. e.g., layers offeeder cells which secrete inductive factors or polymeric layerscontaining inductive factors. For example, a matrigel layer can be usedto induce hematopoietic progenitor cell expansion, as described in theappended examples. Matrigel (Collaborative Research. Inc., Bedford,Mass.) is a complex mixture of matrix and associated materials derivedas an extract of murine basement membrane proteins, consistingpredominantly of laminin, collagen IV, heparin sulfate proteoglycan, andnidogen and entactin was prepared from the EHS tumor as describedKleinman et al, “Basement Membrane Complexes with Biological Activity”,Biochemistry, Vol. 25 (1986), pages 312-318. Other such matrixes can beprovided, such as Humatrix. Likewise, natural and recombinantlyengineered cells can be provided as feeder layers to the instantcultures.

Methods of measuring cell proliferation are well known in the art andmost commonly include determining DNA synthesis characteristic of cellreplication. There are numerous methods in the art for measuring DNAsynthesis, any of which may: be used according to the invention. In anembodiment of the invention. DNA synthesis has been determined using aradioactive label (3H-thymidine) or labeled nucleotide analogues (BrdU)for detection by immunofluorescence.

However, in addition to measuring DNA synthesis, morphological changescan be, and preferably will be relied on as the basis for isolatingresponsive progenitor cell populations. For instance as described in theappended examples we have observed that certain growth factors causeamplification of progenitor cells in ductal explants so as to formstructures that can be easily detected by the naked eye or microscopy.In an exemplary embodiment those progenitor cells which respond togrowth factors by proliferation and subsequent formation of outgrowthsfrom the explant. e.g., buds or blebs can be easily detected. In anotherillustrative embodiment, other structural changes, e.g., changes inoptical density of proliferating cells, can be detected via contrastmicroscopy.

Various techniques may be employed to isolate the activated progenitorcells of treated explant. Preferred isolation procedures for progenitorcells are the ones that result in as little cell death as possible. Forexample, the activated progenitor cells can be removed from the explantsample by mechanical means, e.g., mechanically sheared off with apipette. In other instances, it will be possible to dissociate theprogenitor cells from the entire explant or sub-portion thereof, e.g.,by enzymatic digestion of the explant, followed by isolation of theactivated progenitor cell population based on specific cellular markers,e.g., using affinity separation techniques or fluorescence activatedcell sorting (FACS).

To further illustrate, the examples below demonstrate that ductalexplants contain growth factor responsive progenitor cell types. It isfurther demonstrated that different growth factors can induce/amplifydistinct populations of progenitor cells within the ductal tissueexplant to proliferate. This indicates the presence of specific growthfactor receptors on the surface of distinct progenitor cell populations.This is important because the expression of these receptors marks theprogenitor cell populations of interest. Monoclonal antibodies areparticularly useful for identifying markers (surface membrane proteins.e.g., receptors) associated with particular cell lineages and/or stagesof differentiation. Procedures for separation of the subject progenitorcell may include magnetic separation, using antibody coated magneticbeads, affinity chromatography, and “panning” with antibody attached toa solid matrix. e.g., plate, or other convenient technique. Techniquesproviding accurate separation include fluorescence activated cellsorting, which can have varying degrees of sophistication, e.g., aplurality of color channels, low angle and obtuse light scatteringdetecting channels, impedance channels, etc.

Conveniently, the antibodies may be conjugated with markers, such asmagnetic beads, which allow for direct separation, biotin, which can beremoved with avidin or streptavidin bound to a support, fluorochromes,which can be used with a fluorescence activated cell sorter or the like,to allow for ease of separation of the particular cell type. Anytechnique may be employed which is not unduly detrimental to theviability of the cells.

In an illustrative embodiment, some of the antibodies for growth factorreceptors that exist on the subject progenitor cells are commerciallyavailable (e.g., antibodies for EGF receptors, FGF receptors and/or TGFreceptors), and for other growth factor receptors, antibodies can bemade by methods well known to one skilled in the art. In addition tousing antibodies to isolate progenitor cells of interest, one skilled inthe art can also use the growth factors themselves to label the cells,for example, to permit “panning” processes.

Upon isolation, the progenitor cells of the present invention can befurther characterized in the following manner: responsiveness to growthfactors, specific gene expression, antigenic markers on the surface ofsuch cells and/or basic morphology.

For example, extent of growth factor responsivity, e.g., theconcentration range of growth factor to which they will respond to, themaximal and minimal responses, and to what other growth factors andconditions to which they might respond, can be used to characterize thesubject progenitor cells.

Furthermore, the isolated progenitor cells can be characterized by theexpression of genes known to mark the developing (i.e., stem orprogenitor) cells for liver, pancreas, gall bladder and intestine.

In an illustrative embodiment, the hepatocyte nuclear factor (HNF)transcription factor family, e.g., HNF-1-4 are known to be expressed invarious cell types at various times during liver and pancreasdevelopment. For example, the progenitor cell may express one or moreHNF protein such as HNF1α, HNF1β, HNF3β, HNF3γ, and/or HNF4. ATBF1, aregulator of alphafetoprotein (AFP) gene expression (AFP is expressedearly in liver development and is reexpressed in many liver carcinomas)is believed to be a transient marker of liver stem cells. The LIM typehomeobox genes, such as Islet-1, are also known to be expressed duringliver development. Glut2 is a marker for both early pancreatic and livercells. Certain of the “forkhead” transcription factors, such as fkh-1 orthe like, are understood to be markers in early gut tissue. Likewise,members of the CCAAT-enhancer binding protein (C/EBP) family, such asC/EBP-β, are markers for early liver development. Therefore, expressionof one or more of these genes can be used to further characterize thehepatic progenitor cells.

In another illustrative embodiment, homeodomain type transcriptionfactors such as STF-1 (also known as IPF-1, IDX-1 or PDX) have recentlybeen shown to mark different populations of the developing pancreas.Some LIM genes have also been shown to regulate insulin gene expressionand would also be markers for protodifferentiated β islet cells.Likewise, certain of the PAX genes, such as PAX6, are expressed duringpancreas formation and may be used to characterize certain pancreaticprogenitor cell populations. Other markers of pancreatic progenitorcells include the pancreas specific transcription factor PTF-1, andhXBP-1 and the like. Moreover, certain of the HNF proteins, areexpressed during early pancrease development and may used as markers forpancreatic progenitor cells.

In the intestine, even though there are not very many lineage specifictranscription factors that have been mapped to the gut, the exception isthe Hox B (homeobox genes) gene family (and possibly others) which areregional type markers rather than cell type specific but which can beused to characterize progenitor cells originating form the specificregion. e.g., the gut. Elastase is known to be any early marker ofduodenal development and hence can be a candidate early marker forsubject progenitor cells.

Certain of the subject hematopoietic progenitor cells may becharacterized by expression of such markers as c-kit, CD34 and/or CD33,as well as OV-6, CAM5.2 and/or cytokeratin type 7 or 18.

The subject progenitor cells can also be characterized on the basis ofspecific antigenic markers or other markers that may be expressed on thecell surface, e.g., integrins, lectins, gangliosides, or transporters,or on the basis of specific cellular morphology. All of these techniquesare known and available to the one skilled in the art. For example,certain of the subject hepatic and ductal progenitor cells may expressthe oval cell marker OV-6. Other progenitor cells isolated from theductal microorgan cultures may additionally, or alternatively, expressone or more markers including hematopoietic markers, cytokeratins (suchas type 7 or 19), mucin Span-1, OC.2, OC.3 or γ-glutamyl transpeptidase.Progenitor cells giving rise to pancreatic cells may express such asmarkers as villin and/or tyrosine hydroxylase as well as secrete suchfactors as insulin, glucagon and/or neuropeptide Y.

Once isolated and characterized, the subject progenitor cells can becultured under conditions which allow further differentiation intospecific cell lineages, e.g., hepatic, pancreatic, gallbladder,intestinal, or hematopoietic lineages. This can be achieved through aparadigm of induction that can be developed. For example, the subjectprogenitor cells can be recombined with the corresponding embryonictissue to see if the embryonic tissue can instruct the adult cells tocodevelop and codifferentiate. Alternatively, the progenitor cells canbe contacted with one or more growth or differentiation factors whichcan induce differentiation of the cells. For instance, the cells can betreated with a TGFβ, such as DVR sub-family member.

Furthermore, it has become apparent, from the prolonged viability of theexplanted bile duct fragments in minimal media, that the tissues makingup the ductal explants are themselves producing certain factors. e.g.paracrine and/or autocrine. Accordingly, such conditioned mediagenerated by the explants in culture can be used to further maintain theprogenitor cells in culture subsequent to isolation from the explant.The subject progenitor cells can be cultured in contact with thecorresponding bile duct explant, or in the conditioned media produced bysuch explant.

In another preferred embodiment, the subject progenitor cells can beimplanted into one of a number of regeneration models used in the art,e.g., partial pancreatectomy or streptozocin treatment of a host animal.

Accordingly, another aspect of the present invention pertains to theprogeny of the subject progenitor cells, e.g. those cells which havebeen derived from the cells of the initial explant culture. Such progenycan include subsequent generations of progenitor cells, as well aslineage committed cells. e.g., hepatic, pancreatic or gallbladder cellsgenerated by inducing differentiation of the subject progenitor cellsafter their isolation from the explant, e.g., induced in vitro.

Yet another aspect of the present invention concerns cellularcompositions which include, as a cellular component, substantially purepreparations of the subject progenitor cells, or the progeny thereof.Cellular compositions of the present invention include not onlysubstantially pure populations of the progenitor cells, but can alsoinclude cell culture components. e.g., culture media including aminoacids, metals, coenzyme factors, as well as small populations ofnon-progenitor cells, e.g., some of which may arise by subsequentdifferentiation of isolated progenitor cells of the invention.Furthermore, other non-cellular components include those which renderthe cellular component suitable for support under particularcircumstances, e.g., implantation, e.g., continuous culture.

As common methods of administering the progenitor cells of the presentinvention to subjects particularly human subjects, which are describedin detail herein, include injection or implantation of the cells intotarget sites in the subjects the cells of the invention can be insertedinto a delivery device which facilitates introduction by, injection orimplantation, of the cells into the subjects. Such delivery devicesinclude tubes, e.g., catheters for injecting cells and fluids into thebody of a recipient subject. In a preferred embodiment, the tubesadditionally have a needle, e.g., a syringe through which the cells ofthe invention can be introduced into the subject at a desired location.The progenitor cells of the invention can be inserted into such adelivery device, e.g., a syringe, in different forms. For example, thecells can be suspended in a solution or embedded in a support matrixwhen contained in such a delivery device. As used herein, the term“solution” includes a pharmaceutically acceptable carrier or diluent inwhich the cells of the invention remain viable. Pharmaceuticallyacceptable carriers and diluents include saline aqueous buffer solutionssolvents and/or dispersion media. The use of such carriers and diluentsis well known in the art. The solution is preferably sterile and fluidto the extent that easy syringability exists. Preferably, the solutionis stable under the conditions of manufacture and storage and preservedagainst the contaminating action of microorganisms such as bacteria andfungi through the use of, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Solutions of the invention canbe prepared by incorporating progenitor cells as described herein in apharmaceutically acceptable carrier, or diluent and, as required, otheringredients enumerated above followed by filtered sterilization.

Support matrices in which the progenitor cells can be incorporated orembedded include matrices which are recipient-compatible and whichdegrade into products which are not harmful to the recipient. Naturaland/or synthetic biodegradable matrices are examples of such matrices.Natural biodegradable matrices include plasma clots. e.g., derived froma mammal, and collagen matrices. Synthetic biodegradable matricesinclude synthetic polymers such as polyanhydrides, polyorthoesters, andpolylactic acid. Other examples of synthetic polymers and methods ofincorporating or embedding cells into these matrices are known in theart. See e.g., U.S. Pat. No. 4,298,002 and U.S. Pat. No. 5,308,701.These matrices provide support and protection for the fragile progenitorcells in vivo and are, therefore, the preferred form in which theprogenitor cells are introduced into the recipient subjects.

The present invention also provides substantially pure progenitor cellswhich can be used therapeutically for treatment of various disordersassociated with insufficient functioning of the pancreas or liver.

To illustrate, the subject progenitor cells can be used in the treatmentof a variety of pancreatic disorders both exocrine and endocrine. Forinstance, the progenitor cells can be used to produce populations ofdifferentiated pancreatic cells for repair subsequent to partialpancreatectomy, e.g., excision of a portion of the pancreas. Likewise,such cell populations can be used to regenerate or replace pancreatictissue loss due to, pancreatolysis, e.g., destruction of pancreatictissue, such as pancreatitis, e.g., a condition due to autolysis ofpancreatic tissue caused by escape of enzymes into the substance.

In an exemplary embodiment, the subject progenitor cells can be providedfor patients suffering from any insulin-deficiency disorder. Forinstance, each year, over 728,000 new cases of diabetes are diagnosedand 150.000 Americans die from the disease and its complications; thetotal yearly cost in the United States is over 20 billion dollars(Langer et al. (1993) Science 260: 920-926). Diabetes is characterizedby pancreatic islet destruction or dysfunction leading to loss ofglucose control. Diabetes mellitus is a metabolic disorder defined bythe presence of chronically elevated levels of blood glucose(hyperglycemia). Insulin-dependent (Type 1) diabetes mellitus (“IDDM”)results from an autoimmune-mediated destruction of the pancreaticβ-cells with consequent loss of insulin production, which results inhyperglycemia. Type 1 diabetics require insulin replacement therapy toensure survival. Non-insulin-dependent (Type 2) diabetes mellitus(“NIDDM”) is initially characterized by hyperglycemia in the presence ofhigher-than-normal levels of plasma insulin (hyperinsulinemia). In Type2 diabetes, tissue processes which control carbohydrate metabolism arebelieved to have decreased sensitivity to insulin. Progression of theType 2 diabetic state is associated with increasing concentrations ofblood glucose, and coupled with a relative decrease in the rate ofglucose-induced insulin secretion.

The primary aim of treatment in both forms of diabetes mellitus is thesame namely, the reduction of blood glucose levels to as near normal aspossible. Treatment of Type 1 diabetes involves administration ofreplacement doses of insulin. In contrast, treatment of Type 2 diabetesfrequently does not require administration of insulin. For example,initial therapy of Type 2 diabetes may be based on diet and lifestylechanges augmented by therapy with oral hypoglycemic agents such assulfonylurea. Insulin therapy may be required, however, especially inthe later stages of the disease, to produce control of hyperglycemia inan attempt to minimize complications of the disease, which may arisefrom islet exhaustion.

More recently, tissue-engineering approaches to treatment have focusedon transplanting healthy pancreatic islets, usually encapsulated in amembrane to avoid immune rejection. Three general approaches have beentested in animal models. In the first a tubular membrane is coiled in ahousing that contained islets. The membrane is connected to a polymergraph that in turn connects the device to blood vessels. By manipulationof the membrane permeability, so as to allow free diffusion of glucoseand insulin back and forth through the membrane, yet block passage ofantibodies and lymphocytes, normoglycemia was maintained inpancreatectomized animals treated with this device (Sullivan et al.(1991) Science 252:718).

In a second approach, hollow fibers containing islet cells wereimmobilized in the polysaccharide alginate. When the device was placeintraperitoneally in diabetic animals, blood glucose levels were loweredand good tissue compatibility was observed (Lacey et al. (1991) Science254:1782).

Finally, islets have been placed in microcapsules composed of alginateor polyacrylates. In some cases, animals treated with thesemicrocapsules maintained normoglycemia for over two years (Lim et al.(1980) Science 210:908; O'Shea et al. (1984) Biochim. Biochys. Acta.840:133; Sugamori et al. (1989) Trans. Am. Soc. Artif. Intern. Organs35:791; Levesque et al. (1992) Endocrinology 130:644; and Lim et al.(1992) Transplantation 53:1180). However, all of these transplantationstrategies require a large, reliable source of donor islets.

The pancreatic progenitor cells of the invention can be used fortreatment of diabetes because they have the ability to differentiateinto cells of pancreatic lineage, e.g., β islet cells. The progenitorcells of the invention can be cultured in vitro under conditions whichcan further induce these cells to differentiate into mature pancreaticcells, or they can undergo differentiation in vivo once introduced intoa subject. Many methods for encapsulating cells are known in the art.For example, a source of β islet cells producing insulin is encapsulatedin implantable hollow fibers. Such fibers can be pre-spun andsubsequently loaded with the β islet cells (Aebischer et al. U.S. Pat.No. 4,892,538; Aebischer et al. U.S. Pat. No. 5,106,627; Hoffman et al.(1990) Expt. Neurobiol. 110: 39-44; Jaeger et al. (1990) Prog. BrainRes. 82: 41-46; and Aebischer et al. (1991) J. Biomech. Eng. 113:178-183), or can be co-extruded with a polymer which acts to form apolymeric coat about the β islet cells (Lim U.S. Pat. No. 4,391,909;Sefton U.S. Pat. No. 4,353,888: Sugamori et al. (1989) Trans. Am. Artif.Intern. Organs 35:79-799: Sefton et al, (1987) Biotehnol. Bioeng.29:1135-1143; and Aebischer et al. (1991) Biomaterials 12: 50-55).

Moreover, in addition to providing a source of implantable cells, eitherin the form of the progenitor cell population of the differentiatedprogeny thereof, the subject cells can be used to produce cultures ofpancreatic cells for production and purification of secreted factors.For instance, cultured cells can be provided as a source of insulin.Likewise, exocrine cultures can be provided as a source for pancreatin.

The liver is an organ that is vulnerable to a wide variety of metabolic,circulatory, toxic, microbial, and neoplastic insults, and is,therefore, one of the most frequently injured organs in the body.However, because the function of liver is very complex, syntheticallyrecreating its function is practically impossible. One way of restoringliver function is by whole organ transplantation. Althoughtransplantation of whole liver is often times successful it hasplateaued at about 2200 transplants per year because of donor scarcity.Therefore, alternative treatments concentrate on manipulating thesmallest functional unit of the liver, the individual hepatocyte.

In yet another embodiment, the subject progenitor cells, or theirprogeny, can be used in the treatment various hepatic disorders.Vulnerable to a wide variety of metabolic, circulatory, toxic,microbial, and neoplastic insults, the liver is one of the mostfrequently injured organs in the body. In some instances, the disease isprimarily localized in liver cells. For example, primary liver diseasesinclude hereditary disorders such as Gilbert's Syndrome, Crigler-NajjarSyndrome (either Type I or Type II). Dubin Johnson Syndrome, familialhypercholesterolemia (FH), ornithine transcarbamoylase (OTC) deficiency,hereditary emphysema and hemophilia; viral infections such as hepatitisA. B. and non-A, non-B hepatitis; and hepatic malignancies such ashepatocellular carcinoma. Robbins, S. L. et al. (1984) Pathologic Basisof Disease (W. B. Saunders Company, Philadelphia) pp. 884-942. Moreoften, the hepatic involvement is secondary, often to some of the mostcommon diseases of man, such as cardiac decompensation, disseminatedcancer, alcoholism, and extrahepatic infections. Robbins, S. L. et al.(1984) Pathologic Basis of Disease (W. B. Saunders Company,Philadelphia) pp. 884-942.

Whole liver transplantation, which is the current therapy for a varietyof liver diseases, has been employed to successfully reconstitute LDLreceptors in individuals with FH, thereby lowering serum cholesterol tonormal levels. Whole liver transplantation, however, is limited by thescarcity of suitable donor organs. Li, Q. et al. (1993) Human GeneTherapy 4:403-409; Kay, M. A. et al. (1992) Proc. Natl. Acad. Sci. 89:89-93. In addition to the difficulty in obtaining donor organs, theexpense of liver transplantation, estimated at approximately $200.000 to$300,000 per procedure prohibits its widespread application. Anotherunsolved problem is graft rejection. Foreign livers and liver cells arepoorly tolerated by the recipient and are rapidly destroyed by theimmune system in the absence of immunosuppressive drugs. Li. Q. et al.(1993) Human Gene Therapy 4: 403-409; Bumgardner, G. L. et al. (1992)Transplantation 53: 857-862. While immunosuppressive drugs may be usedto prevent rejection, they also block desirable immune responses such asthose against bacterial and viral infections, thereby placing therecipient at risk of infection.

The hepatic progenitor cells of the invention can be used for treatmentof many liver disorders because they have the ability to differentiateinto cells of hepatic lineage, e.g., hepatocytes. The progenitor cellsof the invention can be cultured in vitro under conditions which canfurther induce these cells to differentiate into mature hepatocytes, orthey can undergo differentiation in vivo once introduced into a subject.Many methods for encapsulating cells are known in the art, as has beendescribed above for pancreatic cells. The hepatic progenitor cells canalso be introduced directly into a subject, as they have the potentialto induce liver regeneration.

Yet another aspect of the present invention provides methods forscreening various compounds for their ability to modulate growth,proliferation or differentiation of distinct progenitor cell populationsfrom bile duct tissue. A micro-organ explant that closely mimics theproperties of a given set of tissue in vivo would have utility inscreening assays in which compounds could be tested for their ability tomodulate one of growth, proliferation or differentiation of progenitorcells in such tissue. Requirements of a reproducible model for screeningmight include consistency in the micro-architecture e.g.epithelial-mesenchymal interactions, and nutritional environment invitro, as well as prolonged viability and proliferation of culturesbeyond 24 hours to observe threshold effects of compounds beingscreened. This level of consistency cannot be achieved in the presenceof undefined media supplements such as sera or tissue extracts that varybetween batches and cannot be adequately controlled. The dependence of amodel on external growth supplements such as growth factors is alsoundesirable as growth factors or hormones may be included among thecompounds to be tested.

In an illustrative embodiment, the ductal explants, which maintain theirmicroarchitecture in culture e.g., they preserve the normalepithelial-mesenchymal architecture that is present in vivo, can be usedto screen various compounds or natural products. Such explants can bemaintained in minimal culture media for extended periods of time (e.g.,for 21 days or longer) and can be contacted with any compound. e.g.,small molecule or natural product, e.g., growth factor to determine theeffect of such compound on one of cellular growth, proliferation ordifferentiation of progenitor cells in the explant. Detection andquantification of growth, proliferation or differentiation of thesecells in response to a given compound provides a means for determiningthe compound's efficacy at inducing one of the growth proliferation ordifferentiation in a given ductal explant. Methods of measuring cellproliferation are well known in the art and most commonly includedetermining DNA synthesis characteristic of cell replication. There arenumerous methods in the art for measuring DNA synthesis, any of whichmay be used according to the invention. In an embodiment of theinvention, DNA synthesis has been determined using a radioactive label(¹H-thymidine) or labeled nucleotide analogues (BrdU) for detection byimmunofluorescence. The efficacy of the compound can be assessed bygenerating dose response curves from data obtained using variousconcentrations of the compound. A control assay can also be performed toprovide a baseline for comparison. Identification of the progenitor cellpopulation(s) amplified in response to a given test agent can be carriedout according to such phenotyping as described above.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

The common bile duct (CBD) is a structure whose developmental origins ispoorly understood. Its primary function is the delivery of bile acidsfrom the liver to the duodenum to aid in the emulsification of food inthe digestive process, 80% of its length traverses through the pancreasand is connected into the pancreatic ductal system through a series ofanastamoses. Through these anastamoses and into the CBD flow thedigestive enzymes secreted by the exocrine pancreas. The relation of theCBD to the liver and pancreas, and its morphology, are thereforeessential to both liver and pancreas normal function.

Since it contributes to both pancreatic and hepatic function, thequestion arises as to whether the the CBD arises during development asprimarily a hepatic structure, a pancreatic structure or both. Littlehas been done to resolve this issue in the past due in part to a lack ofearly markers as well as interest. However, there are a number ofreports in the literature citing circumstantial evidence that the adultanimal retains within the gut system stem cells for both liver andpancreas.

Because the CBD serves a dual hepato-pancreatic function, it might bepossible that it also possesses a dual identity, and might then retainwithin its structure stem cells for both the pancreas and liver. To testthis hypothesis directly we isolated the CBD and its attendant mainducts, and cultured them in vitro as intact duct segments. The goal ofthis work was to study the duct as an intact-physiological unit and todetermine whether resident stem cells could be activated to give rise toeither liver and/or pancreatic derivatives. This unique culture systemallows us the ability to study the interaction of the mesenchyme withthe epithelium, rather than the isolated culture systems of others, andis hence more representative of the in vivo situation.

Using our unique culture system we derived cultures of ductal fragmentsfrom the common bile duct of the adult rat in a combination of matrigeland IGF-1 in serum free conditions induced the formation of liverspecific cell types. The liver identity of the induced cells wasdetermined by immunohistochemical detection of the expression ofalphafetoprotein (AFP), albumin, and ATBF-1, a transcription factorshown to regulate AFP expression. In addition, the formation of redblood cell clusters was observed. The induction of albumin and AFPpositive cell types was specific for the matrigel/IGF-1 combination;culture on plastic or collagen with IGF-1 failed to induce theappearance of these cell types. Likewise, the substitution of IGF-1 witheither TGFα, EGF, IGF-II, PDGF, or FGFβ, all failed to elicit liverformation, even in the presence of matrigel. However, in all of thematrigel conditions tried, we observed the formation of red blood cellclusters. Our results indicate that there exist both liver andhematopoietic stem cells resident in the CBD system, that red blood cellformation can be stimulated by a factor present in matrigel, and thatthe combination of matrigel with IGF-1 can induce the formation of liverspecific cell types.

Preparation of Ductal Explants

6 week old female Spague/Dawley from Taconic were exsanguinated by CO₂.The common bile duct and associated pancreatic ducts was removed andplaced in cold Dulbecco's Minimal Essential Medium (DMEM) supplementedwith 2 mM glutamine and penicillin/streptomycin (100 units/ml). The ductwas then cleaned of associated pancreatic tissue, liver tissue, fat, andblood vessels. The clean and intact duct was sectioned intoapproximately 250 um transverse slices, such that the originalepithelial-mesenchymal microarchitecture was retained, and placed inmedium on ice.

250 μl of reduced growth factor Matrigel (Collaborative Research) wasadded to each well of a 24-well plate and incubated at 37° C. for 30minutes. The plates were then removed and allowed to stand at roomtemperature for 15 minutes. The CBD microorgan explants were placed ontothe matrigel per well and stood at room temperature for 15 minutes toallow the ducts to adhere to the matrigel, 1 ml of DMEM (P/S/L-glut)with or without growth factor addition was added to the wells. Growthfactors used in the studies described below (EGF, TGFα, PDGF, FGFβ,FGFα, IGF-1, and IGF-II) were all obtained from PreproTech. Cultureswere incubated at 37° C. in a 5% CO₂ atmosphere. Cultures were fed oncea week with fresh medium.

Immunohistochemistry

The cultures were fixed overnight at 4° C. in 4% paraformaldehyde (PFA).The cultures were rinsed three times 10 minutes each withphosphate-buffered saline (PBS) prior to addition of 1% periodic acid toremove endogenous peroxidase activity. The cultures were incubated with3% milk in PBS/0.1% Triton X-100 (PBST) for 30 minutes at room,temperature. Primary antibody to alphafetoprotein and albumin (Accurate)were added at 1:250 for 1 hour at room temperature. ATBF-1 was used at1:1000. The tissue was washed 3 times with PBST over a period of 2hours. The secondary biotinylated antibody (Vector) was added at 1:500for 1 hour at room temperature. The tissue was then washed 3 times for 1hour with PBST and incubated with horseradish peroxidase-conjugated toavidin (Vector) for 30 minutes at room temperature. Antibody-binding wasdetected by the addition of DAB/H₂O₂ (Gibco). After termination of thecolorimetric reaction with H₂O, 80% glycerol in PBS was added to clearthe cultures prior to photograph.

Growth Factors

Effectiveness of the growth factors in stimulating proliferation wasjudged by the incorporation of Bromodeoxyuridine (BrdU) into DNA by theresponding cells. Antibodies to BrdU were used to visualize andcharacterize the short term responses (24-48 hr).

The long term response was judged by the ability of these populations ofcells to be grown and expanded in cell culture as a result of specificgrowth factor addition.

Different growth factors (EGF, TGF-α, bFGF, aFGF, IGF-I, IGF-II, VEGFand HGF) were used. In addition to the results for EGF, TGF-α and bFGFdescribed below. IGF-I. IGF-II, VEGF and HGF were demonstrated to causeexpansion of certain subpopulations of the ductal explant.

1. Administration of EGF to the CBD Explant

EGF was administered in three different doses 1 ng/ml, 10 ng/ml and 100ng/ml to the CBD explant. Activation of proliferation as assessed byBrdU labeling occurred with administration of 10 ng/ml of growth factorEGF within a span of 24 hr (FIGS. 1 and 2). There was no differenceobserved between 10 and 100 ng/ml dose. Addition of EGF to the CBDtissue explant resulted in proliferation of distinct cells within theexplant and resulted in clustering of these cells.

2. Administration of TGFα to the CBD Explant

TGFα was administered in the same doses as EGF. Activation ofproliferation as assessed by BrdU labeling occurred with administrationof 100 ng/ml of growth factor TGF-α within a span of 24 hr (FIGS. 1 and2). Unlike EGF, administration of TGF-α to the CBD explant resulted inproliferation of cells throughout the explant.

3. Administration of FGFβ to the CBD Explant

FGFβ was administered in the same doses as the above described growthfactors. Activation of proliferation as assessed by BrdU labelingoccurred with administration of 10 ng/ml of growth-factor FGFβ within aspan of 24 hr (FIGS. 1 and 2). Administration of 10 ng/ml of FGFβresulted in induction of distinct CBD structures to synchronouslydivide, implying organized regulation of proliferative potential andresponse. FIG. 3 depicts in vitro growth and expansion of CBD explant.e.g., formation of outgrowths, e.g., blebs, in response toadministration of FGFβ.

Preliminary long term growth experiments indicate that there does exista large proliferative potential within the CBD explant that can bemaintained in culture for at least 21 days.

Characterization of Expanded Progenitor Cell Populations

Antibodies that recognize transcription factors known to be expressedduring pancreas and liver formation were used to characterize thepopulation distribution in CBDs. STF-1, also known as IPF-1, has beenshown to be critical in pancrease formation; Pax-6 has been shown to becritical in pancreatic endocrine cells; Islet-1 is expressed in earlygut endoderm and in islet tissue; and HNF3β is expressed in early gutendoderm and in liver precursor cells in the endoderm. ATBF-1, Lim1/2were not detected in the CBD. Table 1 shows the average number ofpositive nuclei for each marker per 20× field, or approximately every500 μm duct length. TABLE 1 Scoring of Marker Distribution in the CBDcryostat sections Markers # of nuclei Average STF-1 209 226 170 300Pax-6 26 18 10 18 HNF3β 55 50 46 50 Islet-1 33 38 42 38

HNF3β is an early marker for endoderm formation and is one of theearliest markers expressed by the developing liver. Sections of CBD werestained for the early endoderm and liver marker HNF3β. The common bileduct (CBD) contains cells that are immunopositive for HNF3β. Itsexpression in the CBD is sporadic but specific to the epithelium, asexpected. HNF3β is expressed throughout the CBD and also in the mainpancreatic ducts, although less frequently. Positive cells were oftenfound in clusters of epithelial cells that were branching away from themain lumen of the CBD. HNF3β-expressing cells were found uniformlythroughout the CBD. Because of HNF3β's role as an early endoderm andliver marker in embryonic development, we hypothesized that these. HNF3βexpressing cells might be liver stem cells resident in the adulthepatopancreatic duct system.

If the observed HNF3β expressing cells are indeed liver precursors onemight then use ductal cultures to attempt to in vitro activate theformation of liver-specific cell types and structures. Construction ofsuch a culture system would then allow the study of the signals andinteractions required to induce the liver development program. To thisend we cultured duct fragments that had been transversely cut intoapproximate 250 μm widths.

These were then cultured either on plastic, collagen, directly on feederlayers (STO, C3H10T1/2), or on matrigel (Collaborative Research, Inc.Bedford, Mass.). Matrigel was the only matrix permutation tried thatgave liver formation. Furthermore, the formation of liver structuresonly occurred with the addition of IGF-1. Addition of FGFβ, TGFα, EGF,IGF-II, PDGF-AA all failed to induce the formation of liver structures.

The addition of IGF-1 to duct cultures gave rise to three basic ductcolonies; “Nonresponders” in which there was little observed growth orchange in morphology (FIG. 4A), “N-type” colonies which underwentdramatic changes in morphology to give rise to neuritelike processeswhich were in fact fibroblasts laying end on end (FIG. 4B) and “L-type”colonies which took on a liver-like morphology, with epithelial blebsand the formation of red blood cell clusters (FIG. 4C). This liver-likemorphology resembles that of cultured embryonic liver (data not shown).The number and frequency of the formation of all three colony types isdocumented in the table of FIG. 5.

Because of the morphological similarity between our L-type colonies andcultured embryonic liver, we decided to look for the expression ofliver-specific markers in all three colony types. The markers used forthis analysis were alphafetoprotein (AFP), albumin, and ATBF-1. Allthree have been shown to be specific for early liver. In all casesexamined the only cultures which stained immunopositive for all threemarkers were the L-type colonies.

For example, cultures were stained for the expression of the early livermarker AFP. AFP is expressed within 24 hours of liver formation inembryos, and is lost in adult hepatic tissue. Our data indicatedextensive expression of AFP in IGF-1 treated duct fragments. We also sawloose cells that were highly positive for AFP. These tended to be largecells in diameter. We also observed the formation of duct-likestructures that have arisen in the CBD explants. These duct-likestructures were often surrounded by highly positive AFP staining cells.Some of the duct-like structures appear themselves to be AFP positive,probably due to the fact that AFP is a secreted factor.

Cultures were also stained for the early liver marker ATBF-1. ATBF-1 hasbeen shown to regulate expression of AFP during embryonic liverdevelopment. We observed staining of L-type colonies with the livermarker ATBF-1.

All of the above-cited references and publications are herebyincorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. (canceled)
 2. A cellular composition comprising, as the cellularcomponent, a substantially pure population of viable pancreaticprogenitor cells, which progenitor cells are capable of proliferation ina culture medium and differentiate to pancreatic lineages.
 3. Thecomposition of claim 2, wherein at least 80% of the viable cells in thecomposition are progenitor cells.
 4. The composition of claim 2, whichprogenitor cells are from a mammal.
 5. The composition of claim 4, whichmammal is a transgenic mammal.
 6. The composition of claim 4, whichmammal is a primate.
 7. The composition of claim 6, which mammal is ahuman.
 8. The composition of claim 4, which mammal is a miniature swine.9-13. (canceled)
 14. The composition of claim 2, wherein the progenitorcells express one or more of STF-1; a PAX gene; PTF-1; hXBP-1; an HNFgene; villin; tyrosine hydroxylase; insulin; glucagon; or neuropeptideY.
 15. The composition of claim 14, wherein the progenitor cells expressSTF-1 and PAX6. 16-19. (canceled)
 20. The composition of claim 2, whichprogenitor cells can be maintained in culture for at least about 7 days.21. (canceled)
 22. A cellular composition consisting essentially of, asthe cellular population, viable pancreatic progenitor cells capable ofself-regeneration in a culture medium, which progenitor cellsdifferentiate to members of the pancreatic lineage.
 23. The compositionof claim 22, which progenitor cells are isolated from a hepatic ducttissue, or are the progeny thereof.
 24. The composition of claim 22,which progenitor cells are isolated from cystic duct tissue, or are theprogeny thereof.
 25. The composition of claim 22, which progenitor cellsare isolated from pancreatic duct tissue, or are the progeny thereof.26. The composition of claim 22, which progenitor cells are isolatedfrom common bile duct tissue, or are the progeny thereof.
 27. Thecomposition of claim 22, which progenitor cells are responsive to one ormore growth factor selected from a group consisting of IGF, EGF, TGF,FGF, HGF and VEGF, or orthologous or paralogous factors thereof.
 28. Acellular composition comprising pancreatic progenitor cells, wherein theprogenitor cells are at least 80% pure, are capable of self-regenerationin a culture medium and differentiate to pancreatic lineages.
 29. Thecomposition of claim 28, which progenitor cells are inducible todifferentiate into pancreatic islet cells.
 30. The composition of claim29, which islet cells are pancreatic β islet cells.
 31. The compositionof claim 29, which islet cells are pancreatic α islet cells.
 32. Thecomposition of claim 29, which islet cells are pancreatic δ islet cells.33. The composition of claim 29, which islet cells are pancreatic φislet cells.
 34. The composition of claim 28, wherein the progenitorcells are characterized by expression of STF-1 and PAX6.
 37. Apharmaceutical composition comprising the cellular composition of claim2.
 39. A pharmaceutical composition comprising the cellular compositionof claim
 22. 40. A pharmaceutical composition comprising the cellularcomposition of claim
 28. 41-54. (canceled)
 55. A method for treating adisorder characterized by insufficient insulin activity, in a subject,comprising introducing into the subject a pharmaceutical compositionincluding a substantially pure population of pancreatic progenitor cellsand a pharmaceutically acceptable carrier.
 56. The method of claim 55,wherein the subject is a human.
 57. The method of claim 55, wherein thedisorder is an insulin dependent diabetes.
 58. The method of claim 57,wherein the insulin dependent diabetes is type I diabetes.
 59. Themethod of claim 55, wherein the pancreatic progenitor cells are inducedto differentiate into pancreatic islet cells in the subject.
 60. Themethod of claim 55, wherein the pancreatic progenitor cells are inducedto differentiate into pancreatic β islet cells in culture prior tointroduction into the subject.
 61. The method of claim 55, wherein thepancreatic progenitor cells are characterized by expression of STF-1 andPAX6.
 62. A method for treating a disorder characterized by insufficientliver function, in a subject, comprising introducing into the subject apharmaceutical composition including hepatic progenitor cells and apharmaceutically acceptable carrier.
 63. The method of claim 62, whereinthe subject is a human.
 64. The method of claim 62, wherein the disorderis selected from the group consisting of cirrhosis, hepatitis B,hepatitis C, sepsis or ELAD.
 65. The method of claim 62, wherein thehepatic progenitor cells are induced to differentiate into hepatocytesin the subject.
 66. The method of claim 62, wherein the hepaticprogenitor cells are induced to differentiate into hepatocytes inculture prior to introduction into the subject.
 67. The method of claim62, wherein the hepatic progenitor cells express HNF3, alphafetoprotein(AFP), albumin, and ATBF-1, and differentiate into hepatocytes.